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Description  |
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Immobilization of nucleic acids to solid supports is essential for the
separation and/or identification of specific genes. As has been discussed
in application Ser. No. 511,063, filed July 5, 1983, now abandoned, which
was refiled as application Ser. No. 815,694, filed Jan. 2, 1986, herein
incorporated by reference, the step associated with the gel
electrophoresis for the hybridization and detection of specific genomes
can be eliminated by using a separation probe properly immobilized to a
solid support. In application Ser. No. 511,064, filed July 5, 1983, now
U.S. Pat. No. 4,542,102 issued July 5, 1983 and application Ser. No.
582,503, filed Feb. 22, 1984, now pending, herein incorporated by
reference, we have described two methods of immobilizing nucleic acid
probes to solid supports. The present invention is another method of
immobilizing nucleic acids to solid supports. This method is superior to
the other methods because of the specificity involved and the efficiency
involved in the process. This method has several other advantages. For
example, the method of the invention can be used to immobilize some
primers which can be used to synthesize obligonucleotides on a solid
support by using an enzyme represented in solid supports comprising
cellulose, Sephadex, agarose, nylon, polystyrene, etc., in paper or bead
form. This method eliminates the problem of purification, non-specific
absorption, non-specific coupling and the problem of analysis. The
invention can be described as follows.
A substrate for terminal deoxynucleotidyl transferase or an oligonucleotide
will be immobilized to a solid support. The solid support should be such
that it will not trap the probe and specifically it should have the
porosity to exclude the separation probe and it should be nonreactive when
the separation probe or the immobilized material has to be recovered from
the reaction products. An adenosine triphosphate and related coenzyme
immobilized matrix has been used for affinity chromatography of enzymes
(U.S. Pat. Nos. 4,011,377 and 4,012,283). A similar reaction scheme can be
used to immobilize other nucleic acid phosphates olignonucleotides, and
the immobilized residues can be used for nucleic acid synthesis and probe
immobilization of nucleic acid hybridization. UTP has also been
immobilized by oxidatively reacting the sugar residue. G. Azzar et al,
Anal. Biochem. 142, 518 (1984). The destruction of sugar residue makes it
impossible to use this kind of support for the purpose where nucleoside
phosphates are required for reaction.
The method can be used in several different ways. A typical example can be
given in the following way: 5-allylamino UTP or 5-allylaminodeoxy UTP or
5-allylamino UDP or 5-allylaminodeoxy UDP or 8-hexylamino ATP or deoxy ATP
can be coupled to a solid support via its NH.sub.2 residues and then the
residual nucleoside triphosphate immobilized onto the solid support will
be used as a substrate for terminal deoxynucleotidyl transferase or
polynucleotide phosphorylase to react with the 3' hydroxyl residue of a
DNA or RNA, thus immobilizing the nucleic acid via a phosphordiester
linkage to the immobilized nucleoside phosphates. The immobilized
nucleoside phosphate can be immobilized in such a manner that the 3'
hydroxyl or the 5' phosphate will be available for the enzyme. The
nucleoside diphosphate or triphosphates can be part of a polynucleotide or
oligonucleotide or a mononucleotide.
A purine nucleoside, which may be immobilized in accordance with the
invention, has the following structural formula:
##STR1##
and a pyrimidine nucleoside, which may be immobilized in accordance with
the invention, has the following structural formula:
##STR2##
in which
R and R.sub.1 each independently is --OH, --NH.sub.2, --SH, --COOH or
alkyl, allyl, aryl or alkenyl optionally substituted by --NH.sub.2, --SH,
--OH or
##STR3##
at least one of R and R.sub.1 including an --NH.sub.2, --SH, --OH or
--COOH moiety,
R.sub.2 is --H, --NH.sub.2 or --OH,
Y is H or
##STR4##
n is 1 to 3,
R' is H or OH, and
R" is H, OH or
##STR5##
The immobilization can be done to a solid support via --S--S--,
##STR6##
linkages using known reactions.
Chemical synthesis of deoxyoligonucleotides or ribonucleotides have been
done in a method of stepwise addition of one residue to an immobilized
nucleotide. The methods available are difficult and the purification and
deprotection reactions produce many side products. The present method of
utilization of solid phase enzymatic synthesis eliminates some of these
problems and the probe can be used to label the nucleic acid before it is
even removed from the solid support. The nucleic acid probe can also be
used as a separation probe without removal from the support. An
illustrative example is presented for the synthesis of oligoribonucleotide
and oligodeoxyribonucleotide. The method utilizes an enzyme called T.sub.4
RNA ligase. Other enzymes which are primer independent, e.g., terminal
nucleotidyl transferase can also be used with minor modification of the
method.
The enzyme RNA ligase was first isolated by Silber et al,
Proc.Natl.Acad.Sci., USA, 69, 3009 (1972) from T.sub.4 phage infected E.
coli. It catalyses a reaction between a 3' hydroxyl containing
oligonucleotide and a 5' phosphorylated nucleotide (Snopeck et al,
Biochem.Biophys.Res.Communication 68, 417 (1976)). It has been shown that
3'-, 5'-diphosphates of ribo- and deoxyribo-nucleosides also serve as
donors in the presence of ATP. (England et al, Proc.Natl.Acad.Sci., USA.,
74, 4839 (1977).) The principle of the synthesis can be described as
follows:
##STR7##
EXAMPLE I
Synthesis of a deoxyoligonucleotide specific for the detection of sickle
cell anemia
Step 1: Immobilization of an olignonucleotide acceptor with 3' hydroxyl
available for reaction.
Step 2: Addition of PNP
Step 3: Alkaline phosphatase treatment to dephosphorylate 3' hydroxyl
residue.
Step 4: Repeat of 2 and 3 with the next nucleoside in sequence.
The example is a typical one. There are many variations of the reactions.
In step 1 the immobilization of the acceptor olignonucleotide can be a
deoxyribo- or ribonucleotide or their homopolymers or copolymers. The
linkage can be through a modified nucleoside residue. The linkage can be
single or multiple. The acceptor can be a highly polymerized or tri or
tetramer (the minimum site of the acceptor for the enzyme system). The
bond between the support can be
##STR8##
Step 1: Before immobilization reaction the proper initial acceptor is
chosen such that 3'-hydroxyl will be available and coupling to solid
support can be done under mild condition. Synthesis of the acceptor
5'-dU*pdApdApdApdApdA (U* is a 5-allylamino U residue) is done by a known
phosphoramidite chemistry. Using an Applied Biosystems Model 380A DNA
synthesizer and their chemicals the pentanucleotide (dA).sub.5 is
synthesized. In order to add d*U to (dA).sub.5 the compound 1 is
synthesized. The synthesis scheme is as follows:
##STR9##
EXPERIMENTAL
In the following experimental discussion abbreviations are used as
indicated:
g=gram
HPLC=high performance liquid chromtography
L=liter
mL=milliliter
M=molar
mM=millimolar
N=normal
eq=equivalents
mol=gram molecular formula (moles)
mmol=gram molecular formula.times.10.sup.-3 (millimoles)
aq=aqueous
hr=hour
Infrared (IR) spectra were obtained with a Perkin-Elmer Model 710B or 237
infrared spectrophotometer as solutions in CHCl.sub.3 unless otherwise
noted; the 1602 cm.sup.-1 band of polystyrene film was used as an external
calibration standard. Signals are reported as cm.sup.-1.
Proton magnetic resonance (.sup.1 C NMR) spectra were obtained at 89.55 MHz
using a Varian T-60 spectrometer; spectra were obtained in CDCl.sub.3
solution unless otherwise noted. Chemical shifts are reported in parts per
million downfield from the internal standard tetramethylsilane, unless
otherwise noted.
Carbon-13 magnetic resonance (.sup.13 C NMR) spectra were obtained at 22.5
MHz using a JEOL FX90Q spectrometer with Fourier transform and with full
proton broad-band noise decoupling; spectra were obtained in CDCl.sub.3
solution unless otherwise noted. Carbon shifts are reported in parts per
million downfield from the internal standard tetramethylsilane, unless
otherwise noted.
Phosphorus-31 magnetic resonance (.sup.31 C PNMR) spectra were obtained at
36.21 MHz using a JEOL FX90Q spectrometer; spectra were obtained in
CDCl.sub.3 solution unless otherwise noted. Phosphorus shifts are reported
in parts per million downfield from an external aqueous 15% H.sub.3
PO.sub.4 standard.
Optical rotations were obtained on a Perkin-Elmer Model 141 Polarimeter.
Organic reagents were obtained from Aldrich Chemical Company and were used
without purification, unless otherwise noted. Inorganic reagents were ACS
reagent grade from Fisher Scientific Company or other major vendor.
Reaction solvents were ACS reagent grade. Reagents used in oligonucleotide
synthesis were obtained from Applied Biosystems, Inc. Brine refers to a
saturated aqueous sodium chloride solution.
Thin layer chromtograph (TLC) was performed using silica gel 60F-254 plates
from E. Merck. Column chromatography as performed using E. Merck Silica
Gel 60 (70-230 mesh). All melting points reported are uncorrected.
5-Trifluoroacetamidoallyl-2'-deoxyuridine (compound 8)
A suspension of 5-chloromercuri-2'deoxyuridine (compound 5) (5.56 g; 12
mmol) in HPLC grade methanol (120 ml) was maintained under an inert gas
atmosphere at ambient temperature and treated with
3-trifluoroacetamido-1-propene (7.33 g; 48 mmol; 4 eq) and K.sub.2
PdCl.sub.4 (4.28.g; 1.1 eq). The reaction gradually becomes black and was
allowed to stir for 22 hours. The mixture was treated with H.sub.2 S gas
for several minutes then filtered through Celite, rinsed with MeOH and
evaporated to dryness under reduced pressure from a 80.degree. C. bath to
give a crude semi-solid residue (7.0 g). The residue was chromatographed
on a silica gel column developed with CH.sub.2 Cl.sub.2 /MeOH (5:1). The
band which stained a blue color with modified p-anisaldehyde reagent and
had an Rf=0.51 (CH.sub.3 CN)/MeOH 3:1) was collected and evaporated to
dryness in vacuo to give a colorless foam. The product was crystallized
from a minimum of methanol, filtered, washed with cold CHCl.sub.3 /MeOH
(3:1) and vacuum dried. The mother liquor was worked for a second
crop-total yield 1.01 g (22%). A recrystallization from MeOH afforded the
title compound 8 as analytically pure tiny white needles with
mp=183.degree.-4.degree. C. after drying in vacuo (<1.0 torr) at
64.degree. C. overnight. IR (KBr) cm.sup.-1 3420, 3260, 1718, 1683 (br),
1560, 1478, 1283, 1190, 1102, 1061, 980, 788, 763, 737; 'HNMR
(DMSO-d.sup.6) (Ref. DMSO-d.sup.6) .delta. 2.13 (d of d, J=6 Hz, 2H),
3.59 (br s, 2H). 3.70-3.97 (m, 3H), 4.25 (br s, 1H), 5.06 (br m, 1H), 5.20
(br m, 1H), 6.05-6.65 (m, 4H), 8.01 (s, 1H), 9.60 (br s, 1H); .sup.13 C
NMR (DMSO-d.sup.6) (Ref. DMSO-d.sup.6) ppm 162.05, 155.29, 149.50, 138.05,
124.33, 124.14, 109.96, 87.53, 84.47, 70.23, 61.12, 39.93; [a].sub.D
=+8.01.degree. (c=0.87, MeOH).
Anal. Calcd. for C.sub.14 H.sub.16 N.sub.3 O.sub.6 F.sub.3 : C, 44.33; H,
4.25; N, 11.08. Found: C, 44.19; H, 4.10; N, 10.93.
5-Trifluoroacetamidoallyl-5'-0-(4,4'-dimethoxytrityl)-2'-deoxyuridine
(compound 9)
A solution of compound 8 (0.60 g; 1.58 mmol) in anhydrous pyridine (8 ml)
was maintained under an inert gas atmosphere and treated at ambient
temperature with 4,4'dimethoxytrityl chloride (0.67 g; 1.25 eq). After
stirring for 18 hours the reaction was poured into ice water (70 ml) with
vigorous shaking. On standing 1/3 hour at 0.degree. a gummy solid
separates leaving a nearly clear solution which was decanted. The solid
was washed once with H.sub.2 O (5 ml) then taken up in CH.sub.2 Cl.sub.2
(10 ml), washed once with brine (5 ml) then the CH.sub.2 Cl.sub.2 solution
was dried over K.sub.2 CO.sub.3, filtered and evaporated to dryness in
vacuo to give a brownish foam. The crude product was purified by flash
chromatography on a column of silica gel (Merck, Grade 60, 230-400 mesh,
60 A) (75 g) developed with 4.0% MeOH in CHCl.sub.3 solvent (1.0 L).
Fractions of ca. 20 ml. each were collected in tubes containing pyridine
(10 .mu.l) to inhibit deprotection of the 5'-hydroxyl. Fractions
containing the major product band (RF=0.29; MeOH/CHCl.sub.3 7:93) were
combined, filtered and evaporated to dryness in vacuo to give compound 9
(0.91 g; 85%) as a slightly yellowish foam. A fraction from the center of
the elution band was freed of solvent, taken up in EtoAc, treated with
Norit 211, filtered through Celite and evaporated to dryness under high
vacuum (<1.0 torr) at 64.degree. C. overnight to afford the analytical
sample as a colorless foam with mp=105.degree.-110.degree. C. (dec.). IR
(CHCl.sub.3) cm.sup.-1 3370, 2920, 1715, 1695, 618, 1515, 1470, 1260,
1182, 1045, 842; 'H NMR (CDCl.sub.3) .delta. 2.38 (br m, 2H), 3.25-3.75
(m, 5H), 3.75 (s, 6H), 4.40 (br m, 1H), 4.60 (br s, 1H), 5.39 (d, J=16 Hz,
1H), 6.10-6.55 (m, 2H), 6.70-6.95 (m, 5H), 7.15-7.45 (m, 10H), 7.84 (s,
1H; .sup.13 C NMR (CDCl.sub.3 ) (Ref. CDCl.sub.3) ppm 162.31, 158.74,
157.70, 156.01, 149.70, 144.04, 137.88, 135.65, 135.52, 130.12, 128.11,
127.26, 125.05, 113.48, 111.33, 86.94, 86.68, 85.25, 72.18, 63.60, 55.34,
42.66, 41.42.
Anal. Calcd. for C.sub.25 H.sub.34 N.sub.3 O.sub.8 F.sub.3 : C, 61.67; H,
5.03; N, 6.16. Found: C, 61.47; H, 5.19; N, 5.95.
5-Trifluoroacetamidoaminoallyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyuridine-
3'-O-(N,N-diisopropylaminomethoxy phosphine) (compound 1)
A solution of compound 9 (0.34 g; 0.5 mmol) in anhydrous CH.sub.2 Cl.sub.2
(1.5 ml) maintained under an Argon atmosphere at ambient temperature was
treated first with anhydrous diisopropylethylamine (0.35 ml; 0.259 g; 2
mmol; 4 eq) then dropwize, over 1 minute, with
N,N-diisopropylaminomethoxy-chlorophosphine (compound 10) (0.19 ml; ca.
0.2 g; 2.2 eq). The resultant colorless solution is stirred for 20 minutes
then transferred with EtOAc (20 ml) (EtOAc was previously washed with
saturated aq NaHCO.sub.3 then brine) to a separatory funnel, washed four
times with brine (35 ml each), dried over Na.sub.2 SO.sub.4, filtered and
evaporated to dryness in vacuo to give a colorless glass (0.51 g). This
crude product was taken up in anhydrous benzene (2 ml) and precipitated
into rapidly stirred anhydrous pentane (60 ml) at -78.degree. C. under an
Argon atmosphere. The resulting suspension was filtered, washed with
-78.degree. C. pentane and vacuum dried at <1 torr over KOH overnight to
obtain the title compound 1 (0.38 g; 93%) as a white amorphous powder. IR
(CHCl.sub.3) cm.sup.-1 2965, 1722, 1698, 1618, 1518, 1470, 1262, 1185,
1045, 988, 842; 'H NMR (CD.sub.2 Cl.sub.2) .delta. 0.95-1.30 (m, 12H),
2.20-2.60 (m, 2H), 3.24 and 3.37 (d od d, J=13 Hz, 3H) (P--O--CH.sub.3),
3.20-3.80 (m, 6H), 3.75 (s, 6H), 4.17 (br m, 1H), 4.68 (v br m, 1H), 5.42
(d, J=16 Hz, 1H), 6.15-6.55 (m, 3H), 6.75-6.95 (m, 4H), 7.20-7.50 (m,
10H), 7.79 (s, 1H); .sup.13 C NMR (CD.sub.2 Cl.sub.2) (Ref. CD.sub.2
Cl.sub.2) ppm 162.40, 159.21, 157.78, 149.78, 144.71, 138.34, 136.00,
130.53, 128.71, 128.45, 127.54, 125.66, 125.27, 113.82, 111.48, 87.23,
86.31, 85.60, 55.75, 43.78, 43.20, 42.94, 24.99, 24.60; .sup.31 PNMR
(CD.sub.2 Cl.sub.2) ppm 149.30, 148.87, 14.11 (ca. 12% impurity) 8.18 (ca.
4% impurity).
Attachment of Compound 1 to Oligonucleotides
The 5 unit oligonucleotides dA--p--dA--p--dA--p--dA--p--dA are synthesized
using an Applied Biosystems Model 380A DNA Synthesizer on control pore
glass solid support. Immediately prior to attaching compound 1 to the 5'
end of the oligomer, the 5'-0-(4,4'-dimethoxytrityl) protecting group is
cleaved on the machine with 3% CCl.sub.3 CO.sub.2 H in CH.sub.2 Cl.sub.2
for 90 seconds. The support-bound 5'-deprotected oligomer was washed with
CH.sub.2 CN and dried in an Argon stream. Subsequent steps were performed
without the machine, but using the same chemistry;
1. The support-bound oligomer was removed from the container (column) used
for automated synthesis and transferred to a dry septum-cap vial under an
Argon atmosphere.
2. The bound oligomer was treated with a 20-30 fold excess of 0.5M
1H-Tetrazole in anhydrous CH.sub.3 CN followed immediately with a similar
excess of compound 1 in CH.sub.3 CN. Incubate 30 minutes with gentle
agitation.
3. Pipette off reagents and wash bound oligomer with 3 portions of CH.sub.3
CN.
4. Treat with an excess of I.sub.2 --H.sub.2 O-Lutidine--THF (0.1M:
1:10:40) and agitate for 15 minutes.
5. Pipette off reagent and wash bound oligomer with 4 portions of CH.sub.3
CN.
6. Treat with an excess of Thiophenol-triethylamine-dioxane for 60 minutes.
7. Pipette off reagent and wash the bound oligomer with 4 portions of MeOH.
8. Treat with conc. aq. NH.sub.4 OH for 2 hours at ambient temperature.
(Removes protected oligonucleotide from the support).
9. Add more conc. aq. NH.sub.4 OH and heat at 50.degree. C. overnight.
(Removes all protecting groups except the dimethoxytrityl).
The synthesized oligonucleotide was detritylated with 3% CCl.sub.3 CO.sub.2
H in CH.sub.2 Cl.sub.2 then purified by electrophoresis on polyacrylamide
gel and used to react with an activated support.
This synthesized oligonucleotide has an --NH.sub.2 residue which can be
easily coupled to any activated solid support. N-hydroxysuccinimide ester
of agarose is prepared according to the method described by Cuatrecasas
and Parikh, Biochemistry, 11, 2291 (1972). About 1 gm of moist activated
agarose is shaken gently in 5 ml 0.1M NaHCO.sub.3 buffer (pH 8.6) for 10
minutes at 4.degree. C. Then the oligonucleotide 1 mg/ml in NaHCO.sub.3
buffer is added. The reaction is allowed to proceed for 4 hours at
4.degree. C. with gentle shaking of the solution. Then the unreacted
residues are blocked by adding 1M glycine in the same buffer. The gel is
washed thoroughly with NaHCO.sub.3 buffer (pH 8.6) then with 50 mM HEPES
(pH 8.3). After this washing, the agarose with the immobilized acceptor is
ready for the next step.
Step 2: Addition of pNp:
In order to produce an easily dissociable bond between the acceptor and the
product, the first 2-3 residues used are ribonucleotides. After that
deoxyribonucleotide residues are added. The procedure is identical in both
cases. Only one step addition is described below. The reaction mixture
containing
.about.300 .mu.l immobilized acceptor (step 1)
300 .mu.Moles pNp or deoxy pNp purchased from P.L. Biochemicals
10 ug Bovine serum albumin
100 units of T.sub.4 RNA ligase (P.L. Biochemistry) in 1 ml total volume of
a buffer mixture containing 50 mM HEPES (pH 8.3); 20 mM MgCl.sub.2 ; 3.3
mM dithiothreitol; 8% (v/v) glycerol and 600 .mu.Moles of ATP. The mixture
is incubated for 1 hour at 37.degree. C. After the reaction the mixture is
washed and the solid is separated for the next step. The solution can be
reused for coupling the identical nucleoside.
Step 3: The resin from step 2 is washed and resuspended in HEPES buffer (pH
8.3), then treated with 50 units of alkaline phosphatase (International
Biotechnologies Inc.) at 37.degree. C. for 2 hours and then washed with
HEPES buffer.
Step 4: After the dephosphorylation step of 3, the immobilized residue is
ready for the repeat of the cycle of step 2 and 3 After the desired
synthesis, the oligonucleotide can be removed from the support if a labile
residue is provided, e.g., if the first two residues in step 2 are
ribonucleotide residues and the rest is deoxy residue, digestion with
sodium hydroxide should cleave the phosphodiester linkage at that site. If
an oligoribonucleotide is used in step 1, linkage can be disrupted at that
site in a similar manner or digestion with a ribonuclease can be used. If
RNA synthesis is done, deoxyribose acceptor can be immobilized and can be
cleaved with DNase after the synthesis.
Finally the product is sequenced to establish the efficiency and
specificity of the process.
Following this procedure, an oligonucleotide with 20 residues long is
synthesized and labeled while on the support, by using .sup.32 p labeled
P.sup.C P (from New England Nuclear) the tested for its ability to detect
sickle cell defect by a hybridization technique as has been described by
Conner et al (Proc.Natl.Acad.Sci., USA, 80, 278 (1983)). The sequence
prepared has two ribonucleotide residues and 1% deoxyribonucleotide
residues.
##STR10##
Many other oligonucleotides can be synthesized in an identical fashion.
EXAMPLE 2
Immobilization of separation probe for sickle cell anemia detection via TdT
reaction
About 1 mg of CNBr activated Sepharose C1-4B (prepared by conventional
manner) is washed with 1 mM HCl. 5-allylamino dUTP prepared according to
Lager et. al. Proc. Natl. Acad. Sci, U.S.A. 78, 6633 (1981) is dissolved
in water (concn 1 mg/ml) and kept frozen. After the solid support
activated with cyanogenbromide is washed with HCl they are kept submersed
under 0.1M NaHCO.sub.3 (pH 8.3) for 30 minutes. The buffer is then washed
and fresh solution of NaHCO.sub.3 is added. The washing process is
repeated for 3 times. Then the support is allowed to settle by gravity and
the buffer is removed. Once the solid cake is formed 5-allylamino dUTP is
added .about.10 ml of dUTP solution to 1 ml of the solid, followed by 5 ml
NaHCO.sub.3 buffer. The mixture is shaken by hand and gently shaken in the
cold room for 16 hours. After this time the support is washed to remove
the unreacted AAdUTP. Then 3.times.2 ml 0.2M glycerine solution is added
to block other unreacted active site. The solid is then finally washed
with TdT buffer (0.2M--K-cacodylate buffer (pH 7.2).
The probe which is immobilized is prepared by digestion of a plasmid pSS737
or .beta. pbr322 pst. These plasmids have a pstI segment of human DNA that
includes .beta.-hemoglobin gene. It is easily prepared by published
procedure (obtained from Dr. J. Wilson, Medical College of Georgia
Research Institute and published in Geever et al., Proc. Natl. Acad. Sci.,
U.S.A., 5081 (1981). One sample of the plasmid pSS737 is digested to
completion with DdeI; another sample is digested with Hinf I. The
resulting DNA segments are separated according to size by electrophoresis
in a preparative, low melting temperature agarose gel. The gel is stained
with ethidium bromide for visualization and DNA bands 0.34 Kb from the
HinfI digest and 0.20 Kb from DdeI digest are excised. 0.34 Kg fragment is
immobilized and 0.20 Kb fragment is labeled with 32p by using a
polynucleotide kinase.
100-200 .mu.l of the solid with immobilized A-UTP is suspended in 200 mM
cacodylate buffer and 100 .mu.g of the separation probe, 0.34 Kb. HinfI
segment of pSS 737, application Ser. No. 511,063, supra, in the same
buffer are mixed in an Eppendorf tube. To this MgCl.sub.2 (final conc 4
mM); 1 mM dithiothreitol and 10 units of TdT from BRL are added, the
mixture is incubated at 37.degree. C. for 16 hours. Then the support is
washed with cacodylate buffer.
In order to estimate the capacity of such a supporting material, calf
thymus DNA is used. Using .sup.32 P labeled or .sup.125 I labeled DNA it
is possible to estimate the maximum capacity of the support.
As the hybridization procedure involves single-stranded DNAs, the solid
support upon which the selector probe is immobilized must be pretreated so
that the unknown DNA and the detector probe do not bind to it
indiscriminately. The pretreatment is done with a solution known as
Denhardt's solution (0.2% each of bovine serum albumin, ficoll and
polyvinylpyrrolidone in water), in which, along with some salt and buffer
(e.g., 6.times.SSC, 0.1M Tris, pH 8), the solid is suspended for a few
hours at the temperature to be used for hybridization (e.g., 65.degree.
C.). This solution is then replaced with hybridization medium that
includes denatured sample (unknown) DNA from a patient and denatured
detector probe, and DNA annealing is allowed to proceed for a few hours.
Two representative hybridization conditions are: (i) 6.times.SSC, 0.1M
Tris, pH 8, 65.degree. C., the inclusion of Denhardt's solution being
optional; (ii) 4.times.SSC, 40% formamide, 40.degree. C., .+-.Denhardt's
solution.
After hybridization, DNAs that have not been faithfully base paired to the
selector probe are washed from the support by a series of solutions that
demand extensive annealing for hybrid stability to be maintained. For
example, the solid particles are washed with a large volume of
0.2.times.SSC at 65.degree. C. (at which low salt concentration poorly
base paired hybrids will dissociate), then it is washed with a large
volume of 0.2.times.SSC at room temperature. The particles then are air
dried. If the detector probe is labeled with 32P, the particles are
counted in a scintillation counter. Alternatively, autoradiographic
detection can be done. The extent of radioactivity associated with the
particle is an indication of the disease, sickle cell anemia.
It will be understood that the specification and examples are illustrative
but not limitative of the present invention and that other embodiments
within the spirit and scope of the invention will suggest themselves to
those skilled in the art.
* * * * *
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